Nuclear pore complexes (NPCs) are large proteinaceous assemblies that provide the only known portals for exchanging macromolecules between the nucleus and cytoplasm. This includes the movement of small molecules and the selective, facilitated transport of large proteins and RNAs. Faithful, continuous NPC assembly is key for maintaining normal physiological function and is closely tied to proper cell division. Understanding how NPC biogenesis can be selectively inhibited may be key for designing strategies to inhibit cell growth, for example in oncogenesis. However, the molecular pathway of NPC assembly remains largely undefined. We hypothesize that NPC biogenesis is based on a step-wise mechanism, including a novel vesicle-mediated targeting of NPC-associated proteins to the outer nuclear membrane. The factors that mediate each assembly step are unknown. The long-range goal of this project is to elucidate the molecular sequence of events required for NPC formation. Our specific aims will address the assembly mechanism using the yeast S. cerevisiae as a model. In the first aim, we will identify essential factors required for NPC biogenesis. We have isolated a large collection of temperature sensitive NPC assembly mutants with a fluorescence-based screen. A battery of microscopy and genetic assays will pinpoint mutants with direct defects in new assembly. The second aim builds on our recent discovery that RanGTPase cycle mutants block new NPC assembly by disrupting the targeting of Nup-containing vesicles to the nuclear membrane. We hypothesize that RanGTP is required for both Nup import and for the novel vesicle-mediated step. To investigate this, the accumulated vesicles will be purified and associated proteins tested for assembly roles. Genetic and cell biology strategies will also be used to analyze interactions with Ran. Finally, we will initiate new studies to develop yeast in vitro NPC assembly assay. This is based on our hypothesis that the Nup-containing vesicles are a functional assembly intermediate. Direct tests for the roles of specific proteins in NPC assembly will be conducted and allow for the first time the coupling of genetic, biochemical, and structural analysis of NPC biogenesis in a single system.